ES2380452T3 - Antibodies to inhibit blood clotting and methods of using them - Google Patents

Abstract

Antibody or fragment thereof that binds to human tissue factor ("TF"), in which the antibody or fragment comprises: (i) hypervariable light chain regions CDR1, CDR2, and CDR3, in which: CDR1 comprises the sequence of SEQ ID NO: 5; CDR2 comprises the sequence of SEQ ID NO: 6; and CDR3 comprises the sequence of SEQ ID NO: 7; and (ii) hypervariable heavy chain regions CDR1, CDR2 and CDR3, wherein: CDR1 comprises the sequence of SEQ ID NO: 8; CDR2 comprises the sequence of SEQ ID NO: 9; and CDR3 comprises the sequence of SEQ ID NO: 10.

Description

Antibodies to inhibit blood clotting and methods of using them.

BACKGROUND OF THE INVENTION

one.

 Field of the Invention

[0001] The present invention relates to new antibodies and methods of using antibodies to inhibit blood clotting. In particular, the present invention relates to new antibodies that specifically bind to native human tissue factor with high affinity. The antibodies of the invention are useful for a number of applications, particularly to reduce blood clotting in vivo.

2.

 Background

[0002] Blood coagulation helps homeostasis by minimizing blood loss. In general, blood coagulation requires damage to the vessels, platelet aggregation, coagulation factors and fibrinolysis inhibition. Coagulation factors act through a cascade that relates damage to the vessels with the formation of a blood clot (see generally L. Stryer, Biochemistry, 3rd Ed, WH Freeman Co., New York; and AG Gilman et al., The Pharmacological Basis of Therapeutics, 8th Edition, McGraw Hill Inc., New York, p. 1311-1331).

[0003] There is general agreement that the activation of factor X in factor Xa (FXa) is a critical stage in the blood clotting process. In general, FX is converted to FXa by binding a catalytically active complex that includes "tissue factor" (TF). TF is a cell membrane protein expressed in a controlled manner that binds to factor VII / VIIa to produce the catalytically active complex (TF: VIIa). The blood clot follows the FXa-mediated activation of prothrombin. Blood coagulation can be minimized by inactivating TF in non-native forms that the TF: VIIa complex cannot optimally produce. Excessive formation of FXa is believed to contribute to several thrombosis, including restenosis.

[0004] Thrombosis may be associated with invasive medical procedures, such as cardiac surgery (for example, angioplasty), tummy tuck surgery, arterial surgery, device deployment (for example, a stent or catheter), or endarterectomy. In addition, thrombosis may be accompanied by several thromboembolic disorders and coagulopathies, such as pulmonary embolism (for example, atrial fibrillation with embolization) and disseminated intravascular coagulation, respectively. Manipulation of body fluids can also lead to unwanted thrombi, particularly in blood transfusions or fluid sampling, as well as procedures that involve extracorporeal circulation (for example, cardiopulmonary bypass surgery) and dialysis.

[0005] Anticoagulants are often used to relieve or prevent blood clots associated with thrombosis. Blood coagulation can often be minimized or eliminated by the administration of a suitable anticoagulant or a mixture thereof, including one or more of a coumarin derivative (e.g., warfarin and dicumarol) or a charged polymer (e.g., heparin, hirudin or Bivalirrudine). See, for example, Gilman et al., Supra, R.J. Beigering et al., Ann. Hemathol.,

72: 177 (1996); J.D. Willerson, Circulation, 94: 866 (1996).

[0006] However, the use of anticoagulants is often associated with side effects, such as hemorrhage, reocclusion, "white clot" syndrome, irritation, birth defects, thrombocytopenia and liver dysfunction. Long-term administration of anticoagulants may particularly increase the risk of fatal diseases (see, for example, Gilman et al., Supra).

[0007] Certain antibodies with antiplatelet activity have also been used to relieve several thrombosis. For example, Abciximab is a therapeutic antibody that is usually given to relieve several thromboembolic disorders, such as those that appear from angioplasty, myocardial infarction, unstable angina and coronary artery stenosis. Additionally, Abciximab can be used as a prophylactic agent to reduce the risk of myocardial infarction and angina

(J.T. Willerson, Circulation, 94: 866 (1996); M.L. Simmons et al., Circulation, 89: 596 (1994)).

[0008] Certain anticoagulant antibodies are also known. Particularly, it has been described that certain TF binding antibodies inhibit blood clotting, presumably interfering with the assembly of a catalytically active TF: VIIa complex (see, for example, Jeske et al., SEM in THROM. And HEMO, 22 : 213 (1996); Ragni et al., Circulation, 93: 1913 (1996); European Patent No. 0 420 937 B1; W. Rufet al., Throm. Haemosp., 66: 529 (1991); MM Fiorie et al., Blood, 8: 3127 (1992)).

[0009] However, current TF binding antibodies show significant disadvantages that can minimize their suitability as anticoagulants. For example, current TF binding antibodies do not show sufficient binding affinity for optimal anticoagulant activity. Therefore, for many thrombotic pathologies, to compensate for such ineffective binding affinities, unacceptably high antibody levels must be administered to minimize blood clotting. In addition, current TF binding antibodies do not effectively discriminate between native TF and non-native TF forms, that is, current antibodies do not show sufficient binding specificity. In addition, current TF binding antibodies do not prevent FX from binding to TF and / or the TF: VIIa complex.

[0010] Thus, it would be desirable to have an anticoagulant antibody that binds to native human TF with high affinity and selectivity to thereby inhibit unwanted blood clotting and blood clot formation. It would also be desirable to have said anticoagulant antibody that prevents the binding of Factor X to the TF / VIIa complex.

[0011] Fiore M. M. et al, Blood, vol. 80, no. 12 (December 15), 1992; Pages 3127-3134 describe two monoclonal anti-TF antibodies (TF8-11D12 and TF9-9C3). WO 94/05328 refers to molecules that bind to the tissue factor and alter the TF: VIIa complex. WO 96/40921 provides CDR-grafted antibodies against human tissue factor that maintains the high binding affinity of rodent monoclonal antibodies against tissue factor, but have reduced immunogenicity. WO 89/12463 refers to a therapeutic method and composition for the treatment of myocardial infarction which comprises the administration of a tissue factor protein antagonist and a thrombolytic agent. US 5 437 864 provides a method of inhibiting coagulation of extracorporeal circulation in a subject, which comprises the administration of a therapeutically effective amount of a monoclonal antibody that inhibits the ability of the tissue factor to bind to factor VII / VIIa.

SUMMARY DESCRIPTION OF THE INVENTION

[0012] Antibodies that provide superior anticoagulant activity have been discovered by binding a native human TF with high affinity and specificity. Thus, in one aspect, the present invention provides claim 1. The antibodies of the invention can effectively inhibit blood clotting in vivo. The antibodies of the invention can bind to native human TF, either alone or present in a TF: VIIa complex, effectively preventing the binding of factor X to TF or that complex, and thus reducing blood clotting.

[0013] Preferred antibodies of the invention with monoclonal and specifically bind and bind a predominant conformational epitope to native human TF, whose epitope provides an unexpectedly strong antibody binding site. In fact, preferred antibodies of the invention bind to native human TF at least about 5 times more, more usually at least about ten times more than the binding affinity shown by prior anticoagulant antibodies. Additionally, preferred antibodies of the invention are selective for native human TF and do not bind substantially to non-native or denatured TF. H36.D2.B7 (secreted by the hybridoma ATCC HB-12255) is an especially preferred antibody of the invention.

[0014] Preferred antibodies of the invention bind to TF, so that FX does not bind effectively to the TF / factor VIIa complex, whereby FX does not effectively convert into its activated form (FXa). Preferred antibodies of the invention can inhibit TF function by effectively blocking the binding or access of FX to TF molecules. See, for example, the results of example 3 below.

[0015] Preferred antibodies of the invention neither significantly inhibit the interaction or binding between TF and factor VIIa, or inhibit the activity of the TF complex: factor VIIa with respect to materials other than FX. See, for example, the results of example 4 below.

[0016] The invention also provides nucleic acids encoding antibodies of the invention. The nucleic acid and amino acid sequences (SEQ ID: NOS 1-4) of variable regions of H36.D2.B7 are set forth in Figures 1A and 1B of the drawings.

[0017] In preferred aspects, the antibodies of the invention are for use in the inhibition of blood clotting and blood clot formation and in the reduction of human TF levels.

[0018] In general, the antibodies of the invention will be useful for modulating virtually any biological response mediated by the binding of FX to TF or the TF: VIIa complex, including blood coagulation indicated above, inflammation or other disorders.

[0019] The antibodies of the invention are particularly useful for relieving various thrombosis, particularly to prevent or inhibit restenosis, or other thrombosis after an invasive medical procedure, such as arterial or cardiac surgery (eg, angioplasty). The antibodies of the invention can also be used to reduce or even effectively eliminate blood clotting that arises from the use of medical devices (eg, a catheter, stent or other medical devices). Preferred antibodies of the invention will be compatible with many anticoagulant, antiplatelet and thrombolytic compositions, thus allowing administration in a cocktail format to reinforce or prolong blood clotting inhibition.

[0020] The antibodies of the invention can also be used as an extracorporeal circulating anticoagulant of a mammal, particularly a human subject. In such methods, one or more antibodies are administered to the mammal in an amount sufficient to inhibit blood clotting before or during extracorporeal circulation, such as with cardiopulmonary bypass surgery, organ transplant surgery or other surgeries. prolonged

[0021] The antibodies of the present invention can also be used as carriers for drugs, particularly pharmaceuticals aimed at interaction with a blood clot, such as streptokinase, tissue plasminogen activator (t-PA) or urokinase. Similarly, the antibodies of the invention can be used as a cytotoxic agent by conjugating a suitable toxin to the antibody. Conjugates of the antibodies of the invention can also be used to reduce tissue factor levels in a mammal, particularly a human, by administering to the mammal an effective amount of an antibody of the invention that is covalently bound to a cellular toxin. or an effector molecule to provide complement binding capacity and antibody-dependent cell-mediated cytotoxicity, whereby the antibody conjugate is contacted with cells expressing tissue factor to thereby reduce tissue factor levels in the mammal

[0022] The antibodies of the invention can also be used in in vivo diagnostic methods that include in vivo diagnostic imaging of native human TF.

[0023] The antibodies of the invention can also be used in in vitro assays to detect native TF in a biological sample that includes a biological fluid (eg, plasma or serum) or tissue (eg, a biopsy sample). More particularly, several heterogeneous and homogeneous immunoassays can be used in a competitive or non-competitive format to detect the presence and preferably an amount of native TF in the biological sample.

[0024] Such assays of the invention are very useful in determining the presence or probability of a patient having a blood clot or a blood clot. That is, blood coagulation is normally accompanied by the expression of TF on cell surfaces, such as cells that line the vasculature. In the absence of blood coagulation, TF is not normally expressed. Thus, the detection of TF in a body fluid sample by an assay of the invention will be indicative of blood clotting.

[0025] The antibodies of the invention can also be used to prepare substantially pure native TF, particularly native human TF, from a biological sample. The antibodies of the invention can also be used to detect and purify cells expressing native TF.

[0026] The antibodies of the invention can also be used as a component of a diagnostic kit, for example, to detect and preferably quantify native TF in a biological sample. Other aspects of the invention are described below and are also set forth in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A and 1B show the nucleic acid (SEQ ID NOS: 1 and 3) and amino acid (SEQ ID NOS: 2 and 4) sequences of light chain and heavy chain variable regions of H36.D2.B7 with hypervariable regions (CDRs or Complementarity Determining Regions) underlined (single underline for nucleic acid sequences and double underlined for amino acid sequences).

Figure 2 shows the association (Ka) and dissociation (Kd) constants of anti-tissue factor antibodies as determined by ELISA or BIACore analysis.

Figure 3 shows the inhibition of FX activation mediated by the TF: VIIa complex by preincubation with anti-tissue factor factors.

Figure 4 shows the inhibition of TF / VIIa activity towards the FIBA-specific substrate S-2288 by the tissue factor antibodies.

Figure 5 shows the ability of the H36 antibody to increase prothrombin time (PT) in a coagulation assay initiated by TF.

Figures 6A and 6B graphically show the relationship between FXa formation and the molar ratio of antibody H36.D2 and rhTF. Figure 6A: H36.D2 was pre-incubated with the FT: VIIa complex before adding FX. Figure 6B: H36.D2, TF: VIIa and FX were added simultaneously.

Figure 7 shows the inhibition of TF: VIIa activity by the H36.D2 antibody in a J-82 cell activation assay. Figures 8A and 8B are representations of point transfers showing that the H36.D2 antibody binds to a conformational epitope in rhTF. Lane 1- native rhTF, Lane 2- rhTF native treated with 8M urea, Lane 3- rhTF native treated with 8M urea and 5 mM DTT. In Figure 8A, the transfer was exposed for approximately 40 seconds, while in Figure 8B, the transfer was exposed for 120 seconds.

DETAILED DESCRIPTION OF THE INVENTION

[0028] As described above, preferred antibodies of the invention show substantial affinity for native human TF. In particular, preferred antibodies of the invention show an association constant (Ka, M-1) for native human TF of at least about 1 x 108 as determined by surface plasmon analysis (particularly, BIACore analysis according to the procedures from example 1 below), more preferably at least about 5 x 108 as determined by surface plasmon analysis, even more preferably a Ka (Ka, M-1) for native human TF of at least about 1 x 1010 according to It is determined by surface plasmon analysis. Said substantial antibody binding affinity of the invention clearly contrasts with the much lower affinities of antibodies previously described.

[0029] In this aspect, a fairly low effective concentration of an antibody of the present invention can be used, for example, a relatively low concentration of antibody to inhibit TF function as desired (for example, at least about 95 , 98 or 99 percent inhibition) in an in vitro assay, as described in Example 3 below.

[0030] Preferred antibodies are highly specific for native human TF, and preferably do not bind substantially with non-native TF. Preferred antibodies do not bind substantially to non-native TF or other immunologically unrelated molecules as determined, for example, by a standard point transfer assay (eg, no binding or essentially no binding to non-native TF visually detected by assay point transfer). References of the present invention to "non-native TF" mean a natural or recombinant human TF that has been treated with a chaotropic agent, so that the TF is denatured. Chaotropic agents include a detergent (for example, SDS), urea, combined with dithiothreotol or �-mercaptoethanol; guanidine hydrochloride and the like. The H36, H36.D2 or H36.D2.B7 antibody does not bind substantially to said non-native TF. See, for example, the results of example 8 below and is a point transfer test.

[0031] As described above, the preferred antibodies of the invention will also bind with TF, so that FX does not effectively bind to the TF / factor VIIa complex, whereby FX does not effectively convert into an activated form (FXa). Particularly preferred antibodies of the invention will strongly inhibit FX activity at a TF / factor VIIa complex, for example, an inhibition of at least about 50%, more preferably at least about 80%, and even more preferably by at least about 90% or 95%, even at low concentrations of TF, such as less than about 1.0 nM TF, or even less than about 0.20 nM or 0.10 nM TF, as determined by a standard in vitro binding assay such as that of example 3 below and includes contacting FX with a TF complex: factor VIIa both in the presence (i.e. experimental sample) and in the absence (i.e., control sample) of a antibody of the invention and determine the difference in percentage of the conversion of FX to FXa between the experimental and control samples.

[0032] The antibodies of the invention are preferably substantially pure when used in the methods and assays described. References to an antibody that is "substantially pure" means an antibody or protein that has been separated from naturally occurring components. For example, by using standard immunoaffinity or affinity purification techniques with protein A, an antibody of the invention can be purified from a hybridoma culture by using native TF as an antigen or protein A resin. Similarly, native TF can be obtained in a substantially pure form by using an antibody of the invention with standard immunoaffinity purification techniques. Particularly, an antibody or protein is substantially pure when at least 50% of the total protein (% by weight of the total protein in a given sample) is an antibody or protein of the invention. Preferably, the antibody or protein is at least 60% by weight of the total protein, more preferably at least 75% by weight, even more preferably at least 90% by weight, and most preferably at least 98% by weight of the total material. Purity can be easily analyzed by known methods, such as SDS gel electrophoresis (PAGE), column chromatography (eg, affinity chromatography) in HPLC analysis.

[0033] The nucleic acid sequences (SEQ ID NOS: 1 and 3) and amino acids (SEQ ID NOS: 2 and 4) of a preferred antibody of the invention (H36.D2.B7) are shown in Figures 1A and 1B of the drawings SEQ ID NOS. 1 and 2 are the nucleic acid and amino acid sequences respectively of the light chain variable region, and SEQ ID NOS. 3 and 4 are the nucleic acid and amino acid sequences respectively of the heavy chain variable region, with hypervariable regions (CDR

o Complementary Determinant Regions) underlined in all these sequences. [0034] Additional preferred antibodies of the invention will have a substantial sequence identity with one or both of the light or heavy chain sequences shown in Figures 1A and 1B. More particularly, preferred antibodies include those that have at least about 70 percent homology (sequence identity) with SEQ ID NOS. 2 and / or 4, more preferably about 80 percent or more homology with SEQ ID NOS. 2 and / or 4, even more preferably about 85, 90 or 95 percent or more homology with SEQ ID NOS. 2 and / or 4.

[0035] Preferred antibodies of the invention will have an elevated sequence identity with the hypervariable regions (shown with a double underline in Figures 1A and 1B) of SEQ ID NOS. 2 and 4). Especially preferred antibodies of the invention will have three hypervariable regions of the light chain variable region of H36.D2.B7 (those hypervariable regions shown with the underlined in Figure 1A and are as follows: 1) LASQTID (SEQ ID NO: 5 ); 2) AATNLAD (SEQ ID NO: 6); and 3) QQVYSSPFT (SEQ ID NO: 7)).

[0036] Especially preferred antibodies of the invention will also have three hypervariable regions of the heavy chain variable region of H36.D2.B7 (those hypervariable regions shown with underlining in Figure 1B and are as follows: 1) TDYNVY (SEQ ID NO: 8); 2) YIDPYNGITIYDQNFKG (SEQ ID NO: 9); and 3) DVTTALDF (SEQ ID NO: 10).

[0037] Memory nucleic acids are preferably of sufficient length (preferably at least about 100, 200 or 250 base pairs) to bind the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3 under following moderately stringent conditions (referred to herein as "normal stringency conditions): use of a hybridization buffer comprising 20% formamide in 0.8 M saline buffer / 0.08M sodium citrate (SSC) at a temperature of 37 ° C and that remains attached when it is washed once with that SSC buffer at 37 ° C.

[0038] More preferably, the memory nucleic acids (preferably at least about 100, 200 or 250 base pairs) will be linked to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 3 under the following conditions high stringent (referred to herein as "high ridiculous" conditions): use of a hybridization buffer comprising 20% formamide in 0.9 M saline buffer / 0.09M sodium citrate (SSC) at a temperature of 42 ° C and that it stays together when it is washed twice with that SSC buffer at 42 ° C.

[0039] Memory nucleic acids preferably comprise at least 20 base pairs, more preferably at least about 50 base pairs, and even more preferably a nucleic acid of the invention comprises at least about 100, 200, 250 or 300 base pairs.

[0040] Generally preferred nucleic acids of the invention will express an antibody of the invention showing preferred binding affinities and other properties described herein.

[0041] Preferred nucleic acids of the invention will also have a substantial sequence identity with one or both of the light or heavy chain sequences shown in Figures 1A and 1B. More particularly, the preferred nucleic acids will comprise a sequence that has at least about 70 percent homology (sequence identity) with SEQ ID NOS. 1 and / or 3, more preferably about 80 percent or more homology with SEQ ID NOS. 1 and / or 3, even more preferably about 85, 90 or 95 percent or more homology with SEQ ID NOS. 1 and / or 3.

[0042] Particularly preferred nucleic acid sequences of the invention will have an elevated sequence identity with the hypervariable regions (shown with underlining in Figures 1A and 1B) of SEQ ID NOS. 1 and 3). Especially preferred nucleic acids include those that encode an antibody light chain variable region and that have three sequences encoding hypervariable regions of H36.D2.B7 (those hypervariable regions shown with the underlined in Figure 1A and are as follows: 1 ) CTGGCAAGTCAGACCATTGAT (SEQ ID NO: 11); 2) GCTGCCACCAACTTGGCAGAT (SEQ ID NO: 12); and 3) CAACAAGTTTACAGTTCTCCATTCACGT (SEQ ID NO: 13)).

[0043] Especially preferred nucleic acids also encode an antibody heavy chain variable region and have three sequences encoding hypervariable regions of H36.D2.B7 (those hypervariable regions shown underlined in Figure 1B and are as follows: 1) ACTGACTACAACGTGTAC (SEQ ID NO: 14); 2) TATATTGATCCTTACAATGGTATTACTATCTACGACCA GAACTTCAAGGGC (SEQ ID NO: 15); and 3) GATGTGACTACGGCCCTTGAC TTC (SEQ ID NO: 16)).

[0044] The nucleic acids of the invention are isolated, which means that a given nucleic acid normally constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5 % by weight of the total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of the total nucleic acid present in a given fraction.

[0045] The antibodies of the invention can be prepared by techniques generally known in the art, and usually generated for a purified sample of native TF, usually native human TF, preferably recombinant human tissue factor (rhTF). Truncated recombinant human tissue factor or "rhTF" (composed of 243 amino acids and lacking the cytoplasmic domain) is particularly preferred for generating antibodies of the invention. Antibodies can also be generated from an immunogenic peptide comprising one or more native TF epitopes that are not shown by a non-native TF. References here to "native TF" include said TF samples, including said rhTF. As described above, monoclonal antibodies are generally preferred, although polyclonal antibodies can also be used.

[0046] More particularly, antibodies can be prepared by immunizing a mammal with a purified sample of native human TF, or an immunogenic peptide described above, alone or complexed with a carrier. Suitable mammals include typical laboratory animals, such as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and mice, especially mice, are preferred to obtain monoclonal antibodies. The antigen can be administered to the mammal by a set of suitable routes, such as subcutaneous, intraperitoneal, intravenous, intramuscular injection.

or intracutaneous. The optimal immunization interval, the immunization dose, etc., may vary in relatively wide intervals and can be determined empirically based on this report. The usual procedures involve the injection of the antigen several times for a few months. The antibodies are collected from the serum of the immunized animal by standard techniques and screened to find antibodies specific for native human TF. Monoclonal antibodies can be produced in cells that produce antibodies and these cells are used to generate monoclonal antibodies by standard fusion techniques to form hybridoma cells. See G. Kohler, et al., Nature, 256: 456 (1975). Typically, this involves the fusion of an antibody producing cell with an immortal cell line, such as a myeloma cell, to produce the hybrid cell. Alternatively, monoclonal antibodies can be produced from cells by the method of Huse, et al., Science, 256: 1275 (1989).

[0047] A suitable protocol provides intraperitoneal immunization of a mouse with a composition comprising purified rhTF complex performed over a period of approximately two to seven months. Next, spleen cells can be extracted from the immunized mouse. Immunized mouse serum is analyzed by antibody titers specific for rhTF before spleen cell cleavage. The cleaved mouse spleen cells are then fused to a suitable homogenous (preferably homogeneous) lymphoid cell line having a marker, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency or thymidine kinase (TK-) deficiency. Preferably, a myeloma cell is used as a lymphoid cell line. Myeloma cells and spleen cells are mixed together, for example, at a rate of about 1 to 4 myeloma cells relative to spleen cells. Cells can be fused using the polyethylene glycol (PEG) method. See G. Kohler, et al., Nature, supra. The cloned hybridoma thus develops in a culture medium, for example RPMI-1640. See G. E. More, et al., Journal of American Medical Association, 199: 549 (1967). Hybridomas, developed after the fusion procedure, are screened by, for example, radioimmunoassay or immunoassay with enzymes for secretion of antibodies that specifically bind purified rhTF, for example, antibodies that bind purified rhTF are selected, but not to non-native TF. Preferably, an ELISA is used for screening. Hybridomas that show positive results after such screening can be expanded and cloned by the limiting dilution method. Preferably, additional screening is performed to select antibodies that can bind to rhTF in solution, as well as in a sample of human fluid. The isolated antibodies can be subsequently purified by any suitable immunological technique, including affinity chromatography. The hybridoma culture that produces the preferred particular antibody H36.D2.B7 has been deposited according to the Budapest Treaty with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 10852. The hybridoma culture was deposited with ATCC on January 8, 1997 and was assigned ATCC Access Number HB-12255.

[0048] For human therapeutic applications, it may be desirable to produce chimeric antibody derivatives, for example, antibody molecules that combine a non-human animal variable region and a human constant region, so as to make the antibodies less immunogenic in a human subject. than the corresponding non-chimeric antibody. A variety of types of such chimeric antibodies can be prepared, including, for example, producing chimeras of human variable regions, in which parts of the variable regions, especially conserved regions of the antigen-binding domain, are of human origin and only those Hypervariable regions are of non-human origin. See also the descriptions of chimeric antibodies and their production methods in S.L. Morrison, Science, 229: 1202-1207 (1985); Oi et al., BioTechniques, 4: 214 (1986); Teng et al., Proc. Natl Acad. Sci. U.S.A., 80: 7308-7312 (1983); Kozbor et al., Immunology Today, 4: 7279 (1983); Olsson et al., Meth. Enzymol., 9: 3-16 (1982). Additionally, transgenic mice can be used. For example, transgenic mice have been created that carry repertoires of human antibodies that can be immunized with native human TF. The splenocytes of immunized transgenic mice can then be used to create hybridomas that secrete human monoclonal antibodies that react specifically with native human TF as described above. See, N. Lonberg et al., Nature, 368: 856-859 (1994); L.L. Green et al., Nature Genet., 7: 13-21 (1994); S.L. Morrison, Proc. Natl Acad. Sci. U.S.A., 81: 6851-6855 (1994).

[0049] The antibody nucleic acids of the invention can also be prepared by polymerase chain reaction (see primers described in example 1 below). See, in general, Sambrook et al., Molecular Cloning (2nd ed. 1989). Such nucleic acids can also be synthesized by known methods, for example, the triester transfer method (see Oligonucleotide Synthesis, IRL Press (M.J. Gait, ed., 1984)), or using a commercially available automated oligonucleotide synthesizer. Said nucleic acid of the invention prepared can be used to express an antibody of the invention by known techniques. For example, a nucleic acid encoding an antibody of the invention can be incorporated into a suitable vector by known methods by, for example, the use of restriction enzymes to make cuts in the vector for construction insertion followed by binding. . The vector containing the inserted nucleic acid sequence, operably linked to a promoter sequence, is then introduced into host cells for expression. See, in general, Sambrook et al., Supra. The selection of suitable vectors can be performed empirically based on factors that depend on the cloning protocol. For example, the vector must be compatible with and have the appropriate replicon for the host cell that is used. In addition, the vector must be able to accommodate the inserted nucleic acid sequence. Suitable host cells will include a wide variety of eukaryotic or prokaryotic cells, such as E. coli and the like.

[0050] The molecular weight of the antibodies of the invention will vary depending on several factors, such as the intended use and whether the antibody includes a recombinant conjugated or fused toxin, a detectable pharmaceutical product or label, or the like. In general, an antibody of the invention will have a molecular weight between about 20 to 150 kDa. Such molecular weights can be easily determined by molecular size methods, such as SDS-PAGE gel electrophoresis followed by protein staining or Western blot analysis.

[0051] "Antibody of the invention" or another similar term refers to complete immunoglobulin, as well as immunologically active fragments that bind native TF. Immunoglobulins and immunologically active fragments thereof include an antibody binding site. (ie, an epitope capable of specifically binding to native human TF.) Example antibody fragments include, for example, Fab, F (v), Fab ', F (ab') 2 fragments, "half molecules "obtained by reducing the disulfide bonds of immunoglobulins, single chain immunoglobulins, or other suitable antigen-binding fragments (see, for example, Bird et al., Science, pp. 242-424 (1988); Huston et al. ., PNAS, (USA), 85: 5879 (1988); Webber et al., Mol. Immunol., 32: 249 (1995).) The antibody or immunologically active fragment thereof may be animal (for example, a rodent, such as a mouse or rat), or a chimeric form (see Morrison et al., PNAS, 81: 6851 (1984); Jones et al., Nature, pp. 321, 522 (1986)). Single chain antibodies of the invention may be preferred.

[0052] Similarly, a "nucleic acid of the invention" refers to a sequence that can be expressed to provide an antibody of the invention as specified which means immediately above.

[0053] As described above, the antibodies of the invention can be administered to a mammal, preferably a primate, such as a human, to prevent or reduce thrombosis, such as restenosis, usually in a composition that includes one or more. Non-toxic pharmaceutically acceptable carriers, such as sterile water or saline solution, oils of vegetable origin, and the like. In particular, they can be copolymers of lactide polymer, lactide glycolide or copolymers of polyoxyethylene, polyoxypropylene as excipients for controlling the release of compositions containing antibodies described herein. Other potentially useful delivery systems include ethylene and vinyl acetate particles, osmotic pumps and implantable perfusion systems and liposomes. In general, an anticoagulant composition of the invention will be in the form of a solution or a suspension and will preferably include about 0.01% to 10% (w / v) of the antibody of the present invention, preferably about 0.01% at 5% (w / w) of the antibody. The antibody can be administered as a single active ingredient in the composition, or as a cocktail that includes one or more other anticoagulants (for example, heparin, hirudin, or Bivalirudin), antiplatelets (for example, Abciximab or thrombolytic agents (for example , tissue plasminogen activator, streptokinase and urokinase) Additionally, the antibodies of the invention can be administered before, or after, the administration of one or more anticoagulant, antiplatelet or thrombolytic agents to reinforce or prolong the desired anticoagulant activity.

[0054] As also described above, the antibodies of the invention can be used to reduce the potential blood clotting that arises from the use of medical devices, for example, an internal action device, such as a catheter, a stent , etc. In a preferred method, the device can be treated with an antibody of the invention (for example, as a 1 mg / ml saline solution) before coming into contact with a body fluid. Alternatively, or additionally, an antibody of the invention can be combined with the body fluid in an amount sufficient to minimize blood clotting.

[0055] The therapeutic anticoagulant compositions according to the present invention are suitable for use in parenteral or intravenous administration, particularly in the form of liquid solutions. Such compositions can be conveniently administered in unit doses and can be prepared according to methods known in the pharmaceutical sector. See Remington’s Pharmaceutical Sciences, (Mack Publishing Co., Easton PA, (1980)). By the term "unit dose" is meant a therapeutic composition of the present invention used in a physically discrete unit suitable as a unit dose for a primate, such as a human, each unit containing a predetermined amount of active material calculated to produce the therapeutic effect. desired in association with the diluent or carrier required. The unit dose will depend on a variety of factors including the type and severity of the thrombosis to be treated, the ability of the subject's blood coagulation system to use the antibody, the degree of inhibition or neutralization of the desired FX activation. The precise amounts of the antibody to be administered will typically be guided by the physician's judgment, although the unit dose will generally depend on the route of administration and will be in the range of 10 ng / kg of body weight to 50 mg / kg of body weight per day, more usually in the range of 100 ng / kg body weight up to about 10 mg / kg body weight per day. Suitable guidelines for initial administration in booster administrations are also variable, but are typified by an initial administration followed by repeated doses at intervals of one or more hours by subsequent injection or other administration. Alternatively, continuous or intermittent intravenous infusions can be performed sufficiently to maintain concentrations of at least from about 10 nanomolar to 10 micromolar of the antibody in the blood.

[0056] In some cases, it may be desirable to modify the antibody of the present invention to transmit a desirable biological, chemical or physical property thereto. More particularly, it may be useful to conjugate (i.e. covalently bind) the antibody to a pharmaceutical agent, for example, a fibrinolytic drug, such as t-Pa, streptokinase, or urokinase to provide fibrinolytic activity. Said binding can be performed by various methods, including the use of a binding molecule, such as a heterobifunctional protein crosslinking agent, for example, SPDP, carbodimide, or the like, or by recombinant methods.

[0057] In addition to pharmaceutical products, such as a fibrinolytic agent, an antibody of the invention can be conjugated to a toxin of, for example, plant or bacterial origin, such as diphtheria toxin (ie, DT), toxin shiga, abrin, cholera toxin, ricin, saporin, pseudomonas (PE) exotoxins, ombú or gelonin antiviral protein. Biologically active fragments of said toxins are well known in the art and include, for example, DT A chain and ricin A chain. The toxin can also be an active agent on cell surfaces, such as phospholipases (for example, phospholipase C). As another example, the toxin can be a chemotherapeutic drug, such as, for example, vendesine, vincristine, vinblastine, methotrexate, adriamycin, bleomycin, or cisplatin, or, the toxin can be a radionuclide, such as, for example, iodine. 131, yttrium-90, rhenium-188 or bismuth-212 (see generally, Moskaug et al., J. Biol. Chem., 264: 15709 (1989); 1.

Pastan et al., Cell, 47: 641 (1986); Pastan et al., Recombinant Toxins as Novel Therapeutic Agents, Ann. Rev. Biochem., 61: 331 (1992); Chimeric Toxins Olsnes and Phil, Pharmac. Ther., 25: 355 (1982); published PCT application No. WO 94/29350; published PCT application No. WO 94/04689; and U.S. Patent No. 5,620,939). In addition, as described above, in addition to a toxin, an antibody of the invention can be conjugated to an effector molecule (eg, IgG1 or IgG3) to provide the ability to complement complement and antibody-dependent cell-mediated cytotoxicity. after administration to a mammal.

[0058] Said antibody / cytotoxin conjugate or effector molecule can be administered in a therapeutically effective amount to a mammal, preferably a primate, such as a human, where it is known that the mammal has or is suspected of having tumor cells, cells of the immune system, or endothelial cells capable of expressing TF. Examples of such tumor cells, immune system cells and endothelial cells include malignant tumors of the breast and lung, monocytes and vascular endothelium.

[0059] The antibodies of the invention can also be conjugated to a variety of other pharmaceutical agents in addition to those described above, such as, for example, enzymes, hormones, chelating agents capable of binding to a radionuclide, as well as other proteins and polypeptides. useful for the diagnosis or treatment of the disease. For diagnostic purposes, the antibody of the present invention can be used in a detectable or unlabelled form. For example, a wide variety of markers can be suitably used to detectably label the antibody, such as radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands, such as, for example, haptens and Similar.

[0060] Diagnostic methods are also described including obtaining diagnostic images in vivo [see, for example, A.K. Abbas, Cellular and Molecular Immunology, p. 328 (W.B. Saunders Co. 1991)]. For most imaging applications in vivo, an antibody of the invention can be labeled detectably with, for example, 125I, 32P, 99Tc, or other detectable label, and subsequently administered to a mammal, particularly a human, for a predetermined amount of time sufficient to allow the antibody to come into contact with a desired target. The subject is then screened by known procedures, such as scintigraphic chamber analysis to detect antibody binding. The analysis could help in the diagnosis and treatment of a set of thrombosis, such as those specifically described here. The method is particularly useful when used in conjunction with cardiac surgery, particularly angioplasty, or another surgical procedure where unwanted blood clot formation can take place, to visualize the development or movement of a blood clot.

[0061] The antibodies of the invention can also be used to prepare substantially pure native TF (for example, at least about 90% pure, preferably at least about 96 or 97% pure), particularly native human TF from a biological sample For example, native TF can be obtained as previously described (see, for example, LVM Rao et al., Thrombosis Res., 56: 109 (1989)) and purified by mixing the solution with a solid support comprising the antibody to form a coupling reaction mixture. Example solid supports include a wall of a plate, such as a microtiter plate, as well as supports that include or consist of polystyrene, polyvinyl chloride, a cross-linked dextran, such as Sephadex ™ (Pharmacia Fine Chemicals), agarose, polystyrene particles (Abbott Laboratories), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon and the like. The TF can then be isolated from the solid support in substantially pure form according to standard immunological techniques. See, in general, Harlow and Lane in Antibodies: A Laboratory Manual, CSH Publications, New York (1988) and Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989).

[0062] As described above, the antibodies of the invention can be used to detect native human TF in a biological sample, particularly native TF associated with a blood clot. Example biological samples include blood plasma, serum, saliva, urine, feces, vaginal secretions, bile, lymph, ocular humors, cerebrospinal fluid, cell culture medium and tissue, particularly vascular tissues, such as cardiac tissue. Samples can be properly obtained from a mammal that suffers or is suspected of having a thrombosis, preferably restenosis, associated with, for example, an invasive medical procedure, such as cardiopulmonary bypass surgery; a heart disease, such as myocardial infarction, cardiomyopathy, heart valvular disease, unstable angina, or arthritis fibrillation associated with embolism; coagulopathy, including disseminated intravascular coagulation, deployment of a device, such as a stent or catheter; shock (for example, septic shock syndrome), vascular trauma, liver disease, cardiac stroke, malignant tumors (for example, pancreatic, ovarian carcinoma, or small lung cell), lupus, eclampsia, perivascular occlusive disease and kidney disease.

[0063] For such assays, an antibody of the invention can be detectably labeled with a suitable atom or molecule, for example, radioactive iodine, tritium, biotin or reagent capable of generating a detectable product, such as an anti-idiotypic antibody, bound to an enzyme, such as an enzyme, such �-galactosidase or horseradish peroxidase, or a fluorescent label (eg, fluorescein or rhodamine) according to known methods. After contacting the biological sample with the detectably labeled antibody, any unreacted antibody can be separated from the biological sample, the label (or product) is detected by conventional immunological methods including antibody capture assay, antibody sandwich assay. , RIA, ELISA, immunoprecipitation, immunoabsorption and the like (see Harlow and Lane, supra; Ausubel et al. Supra). Any marker (or product) in excess of that detected in a suitable control sample is indicative of the presence of native TF, more particularly a blood clot, in the biological sample. For example, the antibodies of the invention can be detectably labeled to detect, and preferably quantify, native TF according to standard immunological techniques, such as antibody capture assay, ELISA, antibody sandwich assay, RIA, immunoprecipitation, immunoabsorption, and the like In some cases, particularly when a tissue is used, the immunological technique may include fixing tissue with a reagent known to substantially maintain the conformation of the protein (e.g., diluted formaldehyde). See, in general, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989); Harlow and Lane in Antibodies: A Laboratory Manual, CSH Publications, NY (1988).

[0064] The antibodies of the invention can also be used to detect and purify cells expressing native TF, including fibroblasts, brain cells, immune cells (eg, monocytes), epithelium, as well as certain malignant cells. Preferred cell detection and purification methods include conventional immunological methods (eg, flow cytometry methods, such as FACS and "immunospanning"). Substantially pure populations of cells expressing native TF are useful in clinical and research settings, for example, to establish such cells as cultured cells to screen TF binding antibodies.

[0065] The report also describes analysis and diagnostic kits for the detection of native TF, particularly native human TF, in a test sample, especially a body fluid, such as blood, plasma, etc., or tissue as it has been previously described. A preferred kit includes an antibody of the invention labeled for detection. The diagnostic kit can be used in any immunologically acceptable format, such as an ELISA format to detect the presence or amount of native TF in the biological sample.

[0066] The following non-limiting examples are illustrative of the invention. In the following examples and in other points reference is made to antibodies H36 and H36.D2. These antibodies are the same antibody as H36.D2.B7, but H36 is derived from the parent clone, and H36.D2 is obtained from the primary clone, while H36.D2.B7 is obtained from the secondary clone. No differences between these three clones have been observed with respect to the ability to inhibit TF or other physical properties.

EXAMPLE 1 - Preparation and cloning of anti-rhTF monoclonal antibodies. Monoclonal antibodies against rhTF were prepared as follows:

A. Immunization and reinforcements

[0067] Five female BALB / c mice were immunized with 10 μg of lipidized and purified rhTF in each. The mice were initially sensitized intraperitoneally using Hunter's Titermax adjuvant. Three final reinforcements in 0.85% NaCl were administered. The reinforcements were 2, 5.5, and 6.5 months after initial sensitization. All reinforcements were administered intraperitoneally, except the first that was subcutaneous. The final booster was administered 3 days before the fusion and 20 μg were administered.

B. Fusion of mouse spleen lymphocytes with mouse myeloma cells

[0068] Lymphocytes from the spleen of a BALB / c mouse immunized with rhTF were fused with X63-Ag8.653 mouse myeloma cells using PEG 1500. After exposure to PEG, the cells were incubated for one hour in fetal bovine serum heat inactivated at 37 ° C. The fused cells were then resuspended in RPMI 1640 and incubated overnight at 37 ° C with 10% CO2. The next day the cells were plated using RPMI 1640 and supplemented with macrophage culture supernatant.

C. Development of ELISA

[0069] The plates for the ELISA assay were coated with 100 microliters of recombinant tissue factor (0.25 μg / ml) in a carbonate-based buffer. All steps were performed at room temperature. The plates were blocked with BSA, washed, and then the analysis samples and controls were added. Antigen / antibody binding was detected by incubating the plate with goat anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and then using a substrate system of ABTS peroxidase (Kirkegaad and Perry Laboratories). Absorbance was read on an automatic plate reader at a wavelength of 405 nm.

D. Stabilization of rhTF hybridoma cell lines

[0070] Two weeks after the fusion, hybridoma colonization screening was initiated by specific rhTF ELISA. Screening for new colonies continued for three weeks. Positive clones were analyzed every one or two weeks by continued antibody production until fifteen stable clones were frozen.

E. Primary and secondary cloning

[0071] Cloning by limiting dilution was performed on each of the stable stable hybridomas to obtain primary clones. The cells were thawed, grown in culture for a short period of time, and then diluted from 10 cells / well to 0.1 cells / well. Primary clones were analyzed by anti-rhTF ELISA and expanded and frozen from five to six positive clones.

[0072] The secondary anti-rhTF clone, H36.D2.B7, was obtained from the primary clone, H36.D2, prepared and stored in liquid nitrogen as described above. Four different dilutions, 5 cells / well, 2 cells / well, 1 cell / well, 0.5 cells / well of the primary clone in 96-well microtiter plates were prepared to begin secondary cloning. Cells were diluted in culture media of IMDM tissue containing the following additives: 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, GMS-S at 1%, 0.075% NaHCO3. To determine clones that secrete anti-rhTF antibody, supernatants from five individual wells were removed from the 0.2 cell / well microtiter plate after two weeks of growth and analyzed for the presence of anti-rhTF antibody by ELISA assays. as described above. The five clones showed positive results in the ELISA assay, with H36.D2.B7 being the best antibody producer. All five clones were adapted and expanded in RPMI medium containing the following additives: 10% FBS, 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 1% GMS-S, 0.075% NaHCO3, and 0.013 mg / ml oxalacetic acid. H36.D2.B7 was purified by Protein A affinity chromatography from the cell culture supernatant and analyzed for its ability to inhibit TF: VIIa in an FX activation assay. The results indicated that H36.D2.B7 had the same inhibition as the H36.D2 antibody. All cells were stored in liquid nitrogen.

F. Isolation of total RNA from H36.D2.B7

[0073] 269 μg of total RNA of 2.7 x 105 hybridoma cells H36.D2.B7 were isolated. Total RNA isolation was performed as described in the Qiagen RNeasy Midi Kits protocol. The RNA sample was stored in water at -20 ° C until needed.

G. Synthesis of cDNA and cloning of variable regions of the H36.D2.B7 gene

[0074] To obtain the first cDNA chain, a reaction mixture containing 5 μg of total RNA was prepared isolated as indicated above, JS300 reverse primers (all primers are identified as continued) for the heavy chain (HC) and OKA 57 for the light chain (LC), ARnase inhibitor, dNTP, DTT, and superscript II reverse transcriptase, and incubated at 42 ° C for 1 hour. The reaction tube is then incubated at 65 ° C for 15 minutes to stop transcription. After cooling, five units of ARnase were added H and the reaction was allowed to incubate at 37 ° C for 20 minutes. The cDNA sample was stored at -70 ° C until it was needed.

[0075] A PCR (polymerase chain reaction) was performed separately to clone the variable regions both HC and LC of anti-rhTF, H36.D2.B7 of the cDNA produced as indicated above (in the Figures 1A and 1B describe the nucleic acid and amino acid sequences of these variable regions of HC and LC). Three rounds of PCR were performed. Round 1: PCR was processed for 35 cycles at 96 ° C, 53 ° C and 72 ° C using the JS002 forward primer and JS300 reverse primer for HC. For the LC the primer was used forward JS009 and the OKA 57 reverse primer and PCR was processed for 35 cycles at 96 ° C, 63 ° C and 72 ° C. Round 2: be processed both the HC and LC PCR as in Round 1 with the exception that pMC-18 was used to HC forward primer and pMC-15 for LC forward primer. Round 3: PCR was processed for 30 cycles at 96 ° C, 60-65 ° C and 72 ° C using primers H36HCF and H36HCR for HC. For LC, PCR was processed for 30 cycles at 96 ° C, 58 ° C and 72 ° C using primers H36LCF and H36LCR.

[0076] The following primers were used for cloning of variable regions of H36.D2.B7 of HC and LC.

OKA 57: 5’-GCACCTCCAGATGTTAACTGCTC-3 ’(SEQ ID NO: 17)

[0077] JS300: 5’-GAARTAVCCCTTGACCAGGC-3 ’(SEQ ID NO: 18)

[0078] JS009: 5’-GGAGGCGGCGGTTCTGACATTGTGMTGWCMCARTC-3 ’(SEQ ID NO: 19) JS002: 5’-ATTTCAGGCCCAGCCGGCCATGGCCGARGTYCARCTKCARCARYC-3 ’(SEQ ID NO: 20) pMC-15: 5’-CCCGGGCCACCATGKCCCCWRCTCAGYTYCTKG-3 ’(SEQ ID NO: 21) pMC-18: 5’-CCCGGGCCACCATGGRATGSAGCTGKGTMATSCTC-3 ’(SEQ ID NO: 22) H36HCF:

5’-ATATACTCGCGACAGCTACAGGTGTCCACTCCGAGATCCAGCTGCAGCAGTC-3 ’(SEQ ID NO: 23)

H36HCR:

5’-GACCTGAATTCTAAGGAGACTGTGAGAGTGG-3 ’(SEQ ID NO: 24) H36LCF: 5’-TTAATTGATATCCAGATGACCCAGTCTCC-3 ’(SEQ ID NO: 25) H36LCR: TAATCGTTCGAAAAGTGTACTTACGTTTCAGCTCCAGCTTGGTCC (SEQ ID NO: 26) wherein from SEQ ID NO: 17 to 26 above: K is G or T; M is A or C; R is A or G; S is C or G; V is A, C

or G; W is A or T; Y is C or T.

EXAMPLE 2 - Mabs binding activity of the invention

[0079] The Mabs of the invention were used as prepared in Example 1 above. The rhTF molecule was expressed in E.coli and purified by immunoaffinity chromatography according to standard procedures (see Harlow and Lane, supra, Ausubel et al. Supra). The association (Ka) and dissociation (Kd) constants of Mab were determined by ELISA and surface plasmon resonance assays (i.e., BIACore) (see for example, Harlow and Lane, supra; Ausubel et al. Supra; Altschuh et al., Biochem., 31: 6298 (1992); and the BIAcore procedure described by Pharmacia Biosensor). For BIACore tests, rhTF was immobilized on a biosensor chip according to the manufacturer's instructions. The constants were determined for each Mab at four antibody concentrations (0.125 nM, 0.25 nM, 0.5 nM, and 1 nM). [0080] Protein concentrations were determined by standard assay (M.M. Bradford, Anal. Biochem.,

72: 248 (1976)) using Bovine Serum Albumin as a standard and a commercially available dye reagent (Bio-Rad).

[0081] Figure 2 shows the association and dissociation constants for each anti-rhTF Mab. Mab H36 showed the highest association speed (Ka = 3.1 X 1010 M-1) and the lowest dissociation rate (Kd = 3.2 X 10-11 M) of any of the anti-rhTF Mabs analyzed.

EXAMPLE 3 - FXa specific substrate test

[0082] In general, the experiments described in the present invention were performed using rhTF lipidated with phosphatidylcholine (0.07 mg / ml) and phosphatidylserine (0.03 mg / ml) in a ratio 70/30 p / p in Tris- 50 mM HCl, pH 7.5, 0.1% bovine serum albumin (BSA) for 30 minutes at 37 ° C. A stock solution of preformed TF: VIIa complex was produced by incubating 5 nM of the lipidated rhTF and 5 nM of FVIIa for 30 minutes at 37 ° C. The TF: VIIa complex was aliquotted and stored at -70 until needed. Purified human factors VII, VIIa and FX were obtained from Enyzme Research Laboratories, Inc. The following buffer was used for all FXa and FVIIa assays: 25 mM Hepes-NaOH, 5 mM CaCl 2, 150 mM NaCl, 0 BSA 1%, pH 7.5.

[0083] Mabs were screened for the ability to block the activation of FX to Fxa mediated by TF: VIIa. FX activation was determined in two discontinuous steps. In the first step (FX activation), the conversion of FX to FXa was tested in the presence of Ca + 2. In the second step (Fxa activity test), the activation of FX by EDTA was stopped and the formation of FXa was determined using an FXa-specific chromogenic substrate (S-2222). Chromogens S-2222 and S-2288 (see below) were obtained from Chromogenix (distributed by Pharmacia Hepar Inc.). FX activation was performed in 1.5 ml microcentrifuge tubes by incubation of the reaction mixture with TF: VIIa 0.08 nM, either pre-incubated with an anti-rhTF antibody or a control buffer. The reaction mixture was subsequently incubated for 30 minutes at 37 ° C, then 30 nM FX was added followed by an additional incubation for 10 minutes at 37 ° C. FXa activity was determined in 96-well titration plates. Twenty microliters of sample from step one were extracted and mixed with the same volume of EDTA (500 nM) in each well, followed by the addition of 0.144 ml of buffer and 0.016 ml of S-2222 5mM substrate. The reaction mixture was allowed to incubate for an additional 15-30 minutes at 37 ° C. The reaction mixtures were then stopped with 0.05 ml of 50% acetic acid, after which, the absorbance at 405 nm of each reaction mixture was recorded. Inhibition of TF: VIIa activity was calculated from OD405nm values in experimental (plus antibody) and control (without antibody) samples. In some experiments, an anti-hTF antibody, TF / VIIa, and FX were simultaneously added to detect binding competition. Figure 3 shows that the H36.D2 MAb (in bold) inhibited the activity of TF: / VIIa towards FX to a significantly greater degree (95%) than other anti-rHTF Mabs analyzed.

EXAMPLE 4 - FVIIa specific substrate test

[0084] The Mabs were further screened by a specific FVIIa assay. In this assay, 5 nM lipidated rhTF was first incubated with buffer (control) or 50 nM antibody (experimental) in a 96-well microtiter plate for 30 minutes at 37 ° C, then mixed with purified human FVIIa 5 nM (VT = 0.192 ml), followed by a 30 minute incubation at 37 ° C. Then, eight microliters of a 20 mM stock solution of the specific substrate of FVIIa S-2288 (final concentration, 0.8 mM) was added to each well. Subsequently, the reaction mixture was incubated for one hour at 37 ° C. Next, the absorbance at 405 nm was measured after stopping with 0.06 ml of 50% acetic acid. The percentage inhibition of TF / VIIa activity was calculated from the OD405nm values of the experimental and control samples.

[0085] Figure 4 shows that the H36 antibody did not significantly block TF / VIIa activity towards the S2288 substrate when the antibody was pre-incubated with TF (before the addition of VIIa) or added to TF pre-incubated with VIIa (before add antibody). This indicates that H36 does not interfere with the interaction (binding) between TF and FVIIa, and that H36 does not inhibit the activity of TF: VIIa towards a peptide substrate.

EXAMPLE 5 - Prothrombin Time Test (PT)

[0086] The calcified blood plasma will coagulate within a few seconds after the addition of thromplastine (TF); a phenomenon called "prothrombin time" (PT). A prolonged PT is usually a useful indicator of anticoagulation activity (see, for example, Gilman et al. Supra).

[0087] The H36.D2 antibody was investigated for the ability to affect PT according to standard methods using commercially available human plasma (Ci-Trol Control, Level I obtained from Baxter Diagnostics Inc.). Coagulation reactions were initiated by the addition of lipidated rhTF in the presence of Ca ++. The coagulation time was monitored by an automated coagulation time controller (MLA Electra 800). The PT assays were initiated by injecting 0.2 ml of lipidated rhTF (in a 50 mM Tris-HCl buffer, pH 7.5, containing 0.1% BSA, 14.6 mM CaCl2, 0, 07 mg / ml phosphatidylcholine, and 0.03 mg / ml phosphatidylserine) in plastic double well cuvettes. The cuvettes each contained 0.1 ml of the plasma pre-incubated with 0.01 ml of buffer (control sample) or antibody (experimental sample) for 1-2 minutes. The inhibition of TF-mediated coagulation by the H36.D2 antibody was calculated using a standard TF curve in which the log [TF] was plotted against the clotting time log.

[0088] Figure 5 shows that the H36.D2 antibody substantially inhibits coagulation initiated by TF in human plasma. The H36.D2 antibody significantly increased PT time, showing that the antibody is an effective inhibitor of coagulation initiated by TF (up to about 99% inhibition).

EXAMPLE 6 - FX and the H36.D2 antibody compete for binding to the TF complex: VIIa

[0089] The competition experiments were performed between TF / VIIa, FX and the H36.D2 antibody. Figure 6A illustrates the results of an experiment in which a preformed TF / VIIa complex (0.08 nM) was pre-incubated at 37 ° C for 30 minutes in buffer including 0.02 nM, 0.04 nM, 0.08 nM and 0.16 nM of the H36.D2 monoclonal antibody, respectively. Next, FX (30 nM) was added to the mixture of TF / VIIa and H36.D2 antibody and the mixture was allowed to incubate for an additional 10 minutes at 37 ° C. FX activation was stopped with EDTA as previously described. Thus, the Fxa produced was determined by the specific FXa test described in Example 3 above.

[0090] Figure 6B shows the results of an experiment performed on the same line described above, except that the preformed H36.D2 antibody, TF: VIIA, and FX were added simultaneously to initiate the FX activation assay.

[0091] The data set forth in Figures 6A and 6B show that antibody H36.D2 and FX compete for binding to the preformed TF / VIIa complex.

EXAMPLE 7 - Inhibition of TF activity in cell culture

[0092] J-82 is a human bladder carcinoma cell line (available from ATCC) that abundantly expresses native human TF as a cell surface protein. To see if the H36.D2 antibody could prevent FX from binding to native TF expressed on the cell surface, a J-82 FX activation assay was performed on microtiter plates in the presence of FVII (see DS Fair et al. , J. Biol. Chem., 262: 11692 (1987)). To each well, 2 x 105 cells were added and incubated with 50 ng of FVII, buffer (control sample) or anti-TF antibody (experimental sample) for 2 hours at 37 ° C. Then, each well was gently washed with buffer and 0.3 ml of FX (0.05 mg / ml) was added to each well for 30 minutes at room temperature. In some cases, the antibody was added at the same time as FX to detect binding competition for native TF. After this, 0.05 ml aliquots were extracted and added to new wells in a 96-well titration plate containing 0.025 ml of 100 mM EDTA. FXa activity was determined by a specific FXa assay as described in example 3 above. Inhibition of TF activity on the surface of J-82 cells was calculated from the OD405 nm in the absence (control sample) and presence of the antibody (experimental sample).

[0093] Figure 7 shows that the H36.D2 antibody bound to native TF expressed in J-82 cell membranes and inhibited TF-mediated FX activation. These results indicate that the antibody competes with FX for binding to native TF expressed on the cell surface. Taken with the data in Example 8 below, the results also show that the H36.D2 antibody can bind to a conformational epitope in native TF in a cell membrane.

EXAMPLE 8 - Specific binding of the H36.D2 antibody to native rhTF

[0094] The evaluation of the binding of native and non-native H36.D2 to rhTF was performed by a simplified dot transfer test. Specifically, rhTF was diluted to 30 µg / ml in each of the following three buffers: 10 mM 10 mM Tris-HCl, pH 8.0; 10 mM Tris-HCl, pH 8.0 and 8 M urea; and 10 mM Tris-HCl, pH 8.0, 8 M urea and 5 mM dithiothreitol. Incubation in Tris buffer maintains rhTF natively, while treatment with 8 M urea and 5 nM dithiothreitol produces non-native (denatured) rhTF. Each sample was incubated for 24 hours at room temperature. After incubation, a Millipore Immobilon membrane (7x7cm section) was pre-wetted with methanol, followed by 25 mM Tris, pH 10.4, including 20% methanol. After drying the membranes in air, 0.5 µl, 1 µl, and 2 µl of each sample (30 µg / ml) were applied to the membrane and dried under vacuum. After blocking the membrane by PBS containing 5% skim milk (w / v) and 5% NP-40 (v / v), the membrane was probed with the H36.D2 antibody, followed by incubation with a conjugate of goat anti-mouse peroxidase and IgG (obtained from Jackson ImmunoResearch Laboratories, Inc.). After incubation with Western ECL transfer reagents according to the manufacturer's instructions (Amersham), the membrane was wrapped with a plastic film (Saran Wrap) and exposed to an X-ray film several times.

[0095] Figure 8A shows that Mab H36.D2 binds to a conformational epitope in native TF in the presence of Tris buffer or Tris buffer with 8M urea (lanes 1 and 2). The autoradiogram was exposed for 40 seconds. However, when the native TF was denatured with 8M urea and 5mM DTT, the binding of H36.D2 was significantly reduced or eliminated (lane 3). Figure 8B shows an overexposed autoradiogram showing the residual binding of the H36.D2 antibody to non-native rhTF (ie denatured). Overexposure was approximately 120 seconds. Treatment with 8M urea alone probably resulted in only partial denaturation of the native rhTF, since the two disulfide bonds in TF are not reduced. It is also possible that partially denatured TF can be retracted to the native conformation during a subsequent transfer process when urea is removed. These results also clearly differentiate preferred antibodies of the invention that do not bind denatured TF from previously described antibodies that do not selectively bind a conformational epitope and bind denatured TF (see U.S. Patent 5,437,864 where in Figure 18 Western blot analysis shows denatured TF binding by SDS).

SEQUENCE LIST

[0096]

 (one)

 GENERAL INFORMATION

  (i) APPLICANT: Wong, Hing C.  Jiao, Jin-an  Hope, Snow  Lawrence, Luepschen

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TITLE OF THE INVENTION: ANTIBODIES TO INHIBIT BLOOD COAGULATION AND METHODS OF USING THEMSELVES

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 RECIPIENT: Dike, Bronstein, Roberts & Cushman, LLP

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 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY? A: linear

 (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENTIDE: NO (v) TYPE OF FRAGMENT: (vi) ORIGIN:

 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GACATTCAGA TGACCCAGTC TCCTGCCTCC CAGTCTGCAT CTCTGGGAGA AAGTGTCACC ATCACATGCC TGGCAAGTCA GACCATTGAT ACATGGTTAG CATGGTATCA GCAGAAACCA GGGAAATCTC CTCAGCTCCT GATTTATGCT GCCACCAACT TGGCAGATGG GGTCCCATCA AGGTTCAGTG GCAGTGGATC TGGCACAAAA TTTTCTTTCA AGATCAGCAG CCTACAGGCT GAAGATTTTG TAAATTATTA CTGTCAACAA GTTTACAGTT CTCCATTCAC GTTCGGTGCT GGGACCAAGC TGGAGCTGAA A

60 120 180 240 300 321

 (2) INFORMATION FOR SEQ ID NO: 2:

 (i) FEATURES OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) CHAIN: simple (D) TOPOLOGY? A: linear

 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENTIDE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGIN:

 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Asp Ile Gln Met Thr Gln Be Pro Wing Be Gln Be Wing Be Leu Gly 1 5 10 15 Glu Be Val Thr Ile Thr Cys Leu Wing Be Gln Thr Ile Asp Thr Trp 20 25 30 Leu Wing Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ala Wing Thr Asn Leu Wing Asp Gly Val Pro Ser Arg Phe Be Gly 50 55 60 Be Gly Ser Gly Thr Lys Phe Be Phe Lys Ile Be Ser Leu Gln Wing 65 70 75 80 Glu Asp Phe Val Asn Tyr Tyr Cys Gln Gln Val Tyr Ser Ser Pro Phe 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

(2) INFORMATION FOR SEQ ID NO: 3:

 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 351 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY? A: linear

 (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTISENTIDE: NO

 (v) TYPE OF FRAGMENT: (vi) ORIGIN:

 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAGATCCAGC TGCAGCAGTC TGGACCTGAG CTGGTGAAGC CTGGGGCTTC AGTGCAGGTA TCCTGCAAGA CTTCTGGTTA CTCATTCACT GACTACAACG TGTACTGGGT GAGGCAGAGC CATGGAAAGA GCCTTGAGTG GATTGGATAT ATTGATCCTT ACAATGGTAT TACTATCTAC GACCAGAACT TCAAGGGCAA GGCCACATTG ACTGTTGACA AGTCTTCCAC CACAGCCTTC ATGCATCTCA ACAGCCTGAC ATCTGACGAC TCTGCAGTTT ATTTCTGTGC AAGAGATGTG ACTACGGCCC TTGACTTCTG GGGCCAAGGC ACCACTCTCA CAGTCTCCTC A

60 120 180 240 300 351

 (2) INFORMATION FOR SEQ ID NO: 4:

 (i) FEATURES OF THE SEQUENCE: (A) LENGTH: 117 amino acids (B) TYPE: amino acid (C) CHAIN: simple (D) TOPOLOGY? A: linear

 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENTIDE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGIN:

 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Val Ser Cys Lys Thr Xaa Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Val Tyr Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Ile Thr Ile Tyr Asp Gln Asn Phe 50 55 60 Lys Gly Lys Wing Thr Leu Thr Val Asp Lys Be Ser Thr Thr Wing Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Val Thr Thr Ala Leu Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115

(2) INFORMATION FOR SEQ ID NO: 5:

 (i) FEATURES OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) CHAIN: simple (D) TOPOLOGY? A: linear

 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTISENTIDE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGIN:

 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

 Leu Ala Ser Gln Thr Ile Asp 1 5

(2) INFORMATION FOR SEQ ID NO: 6:

 (i) FEATURES OF THE SEQUENCE:

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 FRAGMENT TYPE: N-terminal

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Wing Wing Thr Asn Leu Wing Asp fifteen

(2) INFORMATION FOR SEQ ID NO: 7:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 9 amino acids

(B)

 TYPE: amino acid

 (C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

TYPE OF MOLECULE: Peptide

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 FRAGMENT TYPE: N-terminal

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 7:

 Gln Gln Val Tyr Ser Ser Pro Phe Thr 15

(2) INFORMATION FOR SEQ ID NO: 8:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 6 amino acids

(B)

 TYPE: amino acid

 (C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

TYPE OF MOLECULE: Peptide

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 FRAGMENT TYPE: N-terminal

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Thr Asp Tyr Asn Val Tyr fifteen

(2) INFORMATION FOR SEQ ID NO: 9:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 17 amino acids

 (B)

 TYPE: amino acid

 (C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

TYPE OF MOLECULE: Peptide

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 FRAGMENT TYPE: N-terminal

(saw)

 ORIGIN:

 (xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Tyr Ile Asp Pro Tyr Asn Gly Ile Thr Ile Tyr Asp Gln Asn Phe Lys 15 10 15 Gly

(2) INFORMATION FOR SEQ ID NO: 10:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 8 amino acids

(B)

 TYPE: amino acid

 (C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

TYPE OF MOLECULE: Peptide

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 FRAGMENT TYPE: N-terminal

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 10:

 Asp Val Thr Thr Ala Leu Asp Phe fifteen

(2) INFORMATION FOR SEQ ID NO: 11:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 21 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CTGGCAAGTC AGACCATTGA T 21

(2) INFORMATION FOR SEQ ID NO: 12:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 21 base pairs

 (B)

 TYPE: nucleic acid

 (C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GCTGCCACCA ACTTGGCAGA T 21

(2) INFORMATION FOR SEQ ID NO: 13:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 28 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 13:

 CAACAAGTTT ACAGTTCTCC ATTCACGT 28

(2) INFORMATION FOR SEQ ID NO: 14:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 18 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 14:

ACTGACTACA ACGTGTAC 18

(2) INFORMATION FOR SEQ ID NO: 15:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 51 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 15:

 TATATTGATC CTTACAATGG TATTACTATC TACGACCAGA ACTTCAAGGG C

(2)

 INFORMATION FOR SEQ ID NO: 16:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 24 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: cDNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(2)

 INFORMATION FOR SEQ ID NO: 17:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 23 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

GATGTGACTA CGGCCCTTGA CTTC 24 (iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GCACCTCCAG ATGTTAACTG CTC

(2)

 INFORMATION FOR SEQ ID NO: 18:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 20 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAARTAVCCC TTGACCAGGC

(2)

 INFORMATION FOR SEQ ID NO: 19:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 35 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 19:

 GGAGGCGGCG GTTCTGACAT TGTGMTGWCM CARTC

(2) INFORMATION FOR SEQ ID NO: 20:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

(TO)

 LENGTH: 45 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

 ATTTCAGGCC CAGCCGGCCA TGGCCGARGT YCARCTKCAR CARYC

(2) INFORMATION FOR SEQ ID NO: 21:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 33 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 21:

CCCGGGCCAC CATGKCCCCW RCTCAGYTYC TKG 33

(2) INFORMATION FOR SEQ ID NO: 22:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 35 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 22:

 CCCGGGCCAC CATGGRATGS AGCTGKGTMA TSCTC 35

(2) INFORMATION FOR SEQ ID NO: 23:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 52 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 23:

 ATATACTCGC GACAGCTACA GGTGTCCACT CCGAGATCCA GCTGCAGCAG TC

(2) INFORMATION FOR SEQ ID NO: 24:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 31 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

 (ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 24:

 GACCTGAATT CTAAGGAGAC TGTGAGAGTG G

(2) INFORMATION FOR SEQ ID NO: 25:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 29 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 25:

TTAATTGATA TCCAGATGAC CCAGTCTCC 29

(2) INFORMATION FOR SEQ ID NO: 26:

(i)

 CHARACTERISTICS OF THE SEQUENCE:

 (TO)

 LENGTH: 45 base pairs

 (B)

 TYPE: nucleic acid

(C)

 CHAIN: simple

(D)

 TOPOLOGY: A: linear

(ii)

 TYPE OF MOLECULE: DNA

(iii) HYPOTHETICAL: NO

(iv)

 ANTISENTIDO: NO

(v)

 TYPE OF FRAGMENT:

(saw)

 ORIGIN:

(xi)

 SEQUENCE DESCRIPTION: SEQ ID NO: 26:

 TAATCGTTCG AAAAGTGTAC TTACGTTTCA GCTCCAGCTT GGTCC

Claims (28)

1. Antibody or fragment thereof that binds to human tissue factor ("TF"), wherein the antibody or fragment comprises:

(i)

 hypervariable light chain regions CDR1, CDR2, and CDR3, in which: CDR1 comprises the sequence of SEQ ID NO: 5; CDR2 comprises the sequence of SEQ ID NO: 6; Y CDR3 comprises the sequence of SEQ ID NO: 7; Y

(ii)

 hypervariable regions of heavy chain CDR1, CDR2 and CDR3, in which: CDR1 comprises the sequence of SEQ ID NO: 8; CDR2 comprises the sequence of SEQ ID NO: 9; Y CDR3 comprises the sequence of SEQ ID NO: 10.

2.

 Antibody or fragment according to claim 1, wherein the antibody or fragment is a monoclonal antibody.

3.

 Antibody or fragment according to claim 1 or claim 2, wherein the antibody or fragment is an antibody chimerical.

Four.

 Antibody or fragment according to claim 1 or claim 2, wherein the antibody or fragment is an antibody humanized

5.

 Antibody or fragment according to any of the preceding claims, wherein the antibody or fragment comprises a variable light chain region that has at least 95% identity in the sequence with the amino acid sequence of SEQ ID NO: 2 and / or a heavy chain variable region that has at least 95% identity in the sequence with the amino acid sequence of SEQ ID NO: 4.

6.

 Antibody or fragment according to claim 5, wherein the antibody or fragment comprises a variable region of light chain having the amino acid sequence of SEQ ID NO: 2 and / or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4.

7.

 Antibody or fragment according to any of the preceding claims, wherein the antibody or fragment comprises A constant human region.

8.

 Antibody or fragment according to any of the preceding claims, wherein the antibody or fragment is a single chain antibody.

9.

 Antibody or fragment according to any of the preceding claims, wherein the binding affinity of the antibody or fragment for a human TF is equal to or greater than that of the monoclonal antibody H36.D2.B7 which is produced by the hybridoma which is deposited with the ATCC under accession number HB-12255.

10.

 Antibody or fragment according to any of the preceding claims, wherein the antibody or fragment has a association constant for human TF of at least about 1 x 1010 M-1.

eleven.

 Antibody or fragment according to any of the preceding claims, wherein the binding affinity of the antibody or fragment for a human TF on denatured human TF is equal to or greater than that of the monoclonal antibody H36.D2.B7 which is produced by the hybridoma that is deposited with the ATCC under accession number HB-12255.

12.

 Antibody that binds to human tissue factor ("TF"), in which the antibody is the H36.D2.B7 monoclonal antibody which is produced by the hybridoma that is deposited with the ATCC under accession number HB-12255.

13.

 Humanized antibody that binds to human tissue factor ("TF"), in which the antibody is a humanized derivative of antibody according to claim 12.

14.

 Purification method of human tissue factor ("TF") from a biological sample, the method comprising putting in Contact the biological sample with an antibody or fragment according to any of claims 1 to 13.

fifteen.

 Method for detecting human tissue factor ("TF") in a biological sample, the method comprising contacting the biological sample with an antibody or fragment according to any one of claims 1 to 13.

16.

 Antibody or fragment according to any of claims 1 to 13, for use in the inhibition of coagulation of the blood in a mammal

17.

 Use of an antibody or fragment according to any of claims 1 to 13, for the manufacture of a medicine to inhibit blood clotting in a mammal.

18.

 Antibody or fragment according to claim 16, or use according to claim 17, wherein the mammal suffers or is suspected of having a thrombosis, or the mammal suffers or is susceptible to restenosis associated with an invasive medical procedure, or the mammal suffers from thromboembolic pathology associated with a cardiovascular disease, an infectious disease, a neoplastic disease, or use of a thrombolytic agent.

19.

 Antibody or fragment according to claim 16, or use according to claim 17, wherein the mammal is a human.

twenty.

 Antibody or fragment according to claim 16, or use according to claim 17, wherein the antibody is administered in combination with an antiplatelet composition, a thrombolytic composition or an anticoagulant composition.

twenty-one.

 Antibody or fragment according to claim 16, or use according to claim 17, wherein the antibody is administered in combination with heparin, hirudin, Bivalirudin, Abciximab, tissue plasminogen activator, streptokinase, or urokinase.

22

 Antibody or fragment according to any of claims 1 to 13, for use as a medicament.

2. 3.

 An isolated nucleic acid encoding an antibody or fragment thereof that binds to human tissue factor ("TF"), in which:

(a) the nucleic acid encodes a variable light chain region and comprises the polynucleotide sequences of SEQ ID NOs: 11, 12, and 13; and (b) the nucleic acid encodes a heavy chain variable region and comprises the polynucleotide sequences of SEQ ID NOs: 14, 15, and 16.

24.

 An isolated nucleic acid according to claim 23, comprising the polynucleotide sequence of SEQ ID NO: 1 and the polynucleotide sequence of SEQ ID NO: 3.

25.

 Nucleic acid encoding an antibody or fragment thereof according to any one of claims 1 to 11.

26.

 Vector comprising the nucleic acid according to any of claims 23 to 25.

27.

 Host cell comprising the vector according to claim 26.

28.

 Host cell comprising (a) a first vector comprising a nucleic acid, in which the nucleic acid encodes a light chain variable region of an antibody or fragment thereof that binds to human tissue factor ("TF") and comprises the polynucleotide sequences of SEQ ID NOs: 11, 12, and 13; and (b) a second vector comprising a nucleic acid, in which the nucleic acid encodes a heavy chain variable region of an antibody or fragment thereof that binds to human tissue factor ("TF") and comprises the sequences of polynucleotides of SEQ ID NOs: 14, 15, and 16.

VARIABLE REGION OF ANTI-FACTOR LIGHT CHAIN